This invention concerns new exendin analogues which can be used in the therapy of diabetes mellitus, processes for their production and pharmaceutical preparations containing them.
A functional connection between the small intestine and exocrine pancreas was proven in the 1960""s after it became possible to accurately determine insulin in plasma. The insulin response after oral glucose administration is much stronger than after intravenous glucose administration even if identical plasma levels of glucose are reached. This xe2x80x9cincretin effectxe2x80x9d is explained by the functional combination of the entero-insular axis. Intestinal hormones are responsible for this effect which are released from the small intestine after meals, circulate in the plasma at increased measurable levels and amplify glucose-induced insulin release. In addition to the classical incretin hormone gastric inhibitory polypeptide I (GIP), glucagon-like peptide. 1 (GLP-1) is nowadays of primary interest. In a relatively short time GLP-1 has matured from being the physiologically most interesting incretin hormone candidate to a potential alternative for the treatment of diabetes mellitus type II. The present invention describes new substances which imitate the effect of the naturally occurring GLP-1 molecule. These new substances are characterized by an increased stability while maintaining efficacy.
Infusion and subcutaneous injection of GLP-1 cause a considerable increase of insulin secretion and an inhibition of glucagon release in patients with diabetes mellitus type II (Gutniak, M. (1992); Kreymann, B. (1987); Nathan, D. M. (1992); Nauck, M. A. (1993a and b)). Both are of therapeutic interest and are involved in the blood sugar lowering effect of GLP-1: insulin promotes glucose uptake by its target tissue and inhibits gluconeogenesis. Furthermore GLP-1 analogues would be expected to increase glucose uptake in the periphery. The inhibition of glucagon secretion must be regarded as an indirect GLP-1 effect since glucagon-producing A cells express no GLP-1 receptors (Komatsu, R. (1989)). On the contrary, the increased insulin and somatostatin release appear to be the decisive factor. Both hormones are known as inhibitors of glucagon release.
Two molecular mechanisms certainly contribute to the GLP-1-induced insulin release in diabetes mellitus type II. In addition to directly amplifying the glucose-induced insulin release, GLP-1 sensitizes a subgroup of B cells towards the key stimulus xe2x80x9cglucosexe2x80x9d (Fehmann, H. C. (1991)) and possibly also towards further stimuli so that overall more B cells secrete insulin. This prizing affect is the most likely explanation for the fact that GLP-1 leads to a prolonged release of insulin despite its relatively short plasma half-life.
This effect depends on increased glucose levels ( greater than 108 mg/dl) (Gxc3x6ke, R. (1993a)). It distinguishes GLP-1 fundamentally from the sulfonylureas which influence insulin secretion independently of the plasma level of glucose. If the glucose value decreases below 108 mg/dl, the insulin secretion dries up even with an intravenous infusion of GLP-1. Hence hypoglycaemias would be hardly expected when GLP-1 is used therapeutically. In fact they have also not been described in the previous clinical studies. However, the pharmacokinetic properties of GLP-1 are problematic. The duration of action is limited due to its very short half-life.
From a therapeutic point of view the synthesis of stable and strongly effective GLP-1 peptide analogues is in any case desirable. Peptide analogues have now been synthesized based on the molecule exendin that was originally isolated from the venom of lizards with the aim of developing improved therapeutic agents that are stable towards degradation with an increased duration of action for the treatment of diabetes mellitus. These peptides have the same pharmacological effect as GLP-1, but surprisingly have a considerably longer half-life.
The new peptide sequences described as the subject matter of the invention have an effect on insulin synthesis and insulin release and an action on the insulin effect especially the uptake of glucose into the target tissues, muscle and fat tissue as well as emptying of the stomach.
The present invention is based on the sequence of exendin-3 and exendin-4, peptides which were isolated from the secretory product of Heloderma horridum or Heloderma suspectum (Eng. J. et al. (1990, 1992)). The amino acid sequence and effect of the two peptides on the pancreas has already been published by several authors (Eng. J. et a. (1990); Raufman, J. P. (1992); Gxc3x6ke, R. (1993b); Thorens, B. (1993)). The subject matter of this invention are new truncated exendin fragments which comprise the amino acid sequences of exendin-3-(1-30) or exendin-4-(1-30) in which the C-terminal end of these sequences can be shortened by up to 3 amino acids, preferably by at most 1 amino acid, and the N-terminal end can be shortened by up to 2 and preferably at most 1 amino acid. Surprisingly these exendin fragments are biologically active although the amino acid sequence is shortened. Shortened amino acid sequences are more economical to produce than relatively longer sequences. Hence, peptide fragments with the following sequences are particularly preferred; especially peptide fragments that are based on exendin-3-(1-30) (SEQ ID NO.1):
SEQ ID NO:1 based on exendin-3
SEQ ID NO:2 based on exendin-4
in which the amino acids at position 1, 2, 28, 29 or 30 can be part of the sequence depending on the desired chain length. The peptides are numbered through from the N-terminus to the C-terminus. X1 denotes a proteogenic or non-proteogenic amino acid apart from glycine. Exendin and exendin analogues with a chain length of 1-27 are preferred and especially those with a chain length of 1-30.
The carboxyl group COR3 of the amino acid at the C-terminal end can be present in a free form (R3=OH) or in the form of a physiologically tolerated alkaline or alkaline earth salt such as e.g. a sodium, potassium or calcium salt. The carboxyl group can also be esterified with primary, secondary or tertiary alcohols such as e.g. methanol, branched or unbranched C1-C6-alkyl alcohols, in particular ethyl alcohol or tert. butanol. The carboxyl group can, however, also be amidated with primary or secondary amines such as ammonia, branched or unbranched C1-C6 alkylamines or C1-C6 di-alkylamines, in particular methylamine or dimethylamine.
The amino group of the amino acid NR1R2 at the N-terminal end can be present in a free form (R1, R2=H) or in the form of a physiologically tolerated salt such as e.g. a chloride or acetate. The amino group can also be acetylated with acids so that R1=H and R2=acetyl, trifluoroacetyl, adamantyl or be present in a form protected by conventional amino protecting groups of peptide chemistry such as e.g. Fmoc, Z, Boc, Alloc or be N-alkylated in which R1 and/or R2=C1-C6 alkyl or C2-C8 alkenyl or C7-C9 aralkyl.
Alkyl residues are understood as straight-chained, branched or optionally ring-shaped alkyl residues, preferably methyl, ethyl, isopropyl and cyclohexyl.
All exendin fragments can be present as completely or partially protected derivatives.
A further subject matter of this invention are exendin fragments with the above-mentioned properties and sequence lengths in which at least one but at most eleven of the modifications listed under (a) to (p) have been additionally carried out. Exendin fragments are preferred which have at most nine and particularly preferably those which have at most five of the modifications listed under (a) to (p).
(a) The xcex1-amino acid in position 1 is D-His, Ala, D-Ala, Gly, Lys or D-Lys of which Ala, Gly or Lys are particularly preferred; or
(b) the xcex1-amino acid in position 2 is Ser, D-Ser, Thr, D-Thr, Gly, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, preferably Ser, Thr, Gly, Ala, Val, Ile or Leu; or
(c) the xcex1-amino acid in position 3 is Glu, D-Glu, Asp, D-Asp, Ala or D-Ala, preferably Glu, Asp or Ala; or
(d) the amino acid in position 4 is Ala, D-Ala or xcex2-Ala, preferably Ala; or
(e) the xcex1-amino acid in position 5 is Ser, Tyr or Ala; or
(f) the xcex1-amino acid in position 6 is Ala, Ile, Val, Leu, Cha or Tyr, preferably Ala, Ile, Val, Leu or Tyr; or
(g) the xcex1-amino acid in position 7 is Ala, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr, preferably Ala, Tyr or Ser; or
(h) the xcex1-amino acid in position 8 is Ala, Tyr or Thr; or
(i) the xcex1-amino acid in position 9 is Ala, D-Ala, Glu, D-Glu or D-Asp, preferably Ala or Glu; or
(j) the amino acids in position 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29 are independently of one another a proteinogenic or non-proteinogenic D- or L-amino acid, preferably a proteinogenic L-amino acid; or
(k) the xcex1-amino acid in position 13 is a neutral L-amino acid, preferably a neutral proteinogenic L-amino acid; or
(l) the xcex1-amino acid in position 14 is, for the purposes of stabilization, replaced by a neutral L- or D-amino acid, apart from L-leucine, preferably by Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val or D-Val, wherein Ile, Val or Ala are particularly preferred; or
(m) the xcex1-amino acid in position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D-Val, L-Cha, D-Cha, xcex2-1-Nal, xcex2-2-Nal or xcex2-1-D-Nal, wherein Tyr, Leu or Val are preferred; or
(n) the xcex1-amino acid in position 23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D-Cha, Tyr, D-Tyr, Phe or D-Phe, wherein Leu, Val, Tyr or Phe are preferred; or
(o) the xcex1-amino acid in position 25, 26 or 27 is a neutral L- or D-amino acid, preferably a neutral, proteinogenic L-amino acid; or
(p) the xcex1-amino acid in position 30 is a proteinogenic or non-proteinogenic D- or L-amino acid apart from glycine, preferably Arg, D-Arg, Tyr or D-Tyr, Arg or Tyr are particularly preferred.
Among the new exendin fragments, those are particularly preferred which contain the amino acid leucine at position 10 and/or the amino acid valine at position 19, the amino acid isoleucine or alanine instead of methionine at position 14 and arginine at position 30 in addition to the already mentioned properties and sequence lengths. Those modifications of exendin fragments are also particularly preferred in which, in addition to the particularly preferred amino acids at positions 10, 14, 19 and 30, one of the 20 known proteinogenic L-amino acids is located at position 2.
Preferred exendin analogues have a substitution at position 3 or 14, particularly preferably at position 2 and especially preferably the exendin analogues only contain proteinogenic amino acids.
In addition to new shortened and stabilized exendin-3 and exendin-4 analogues, the invention also concerns processes for producing these analogues in which the analogues are prepared in a solid phase synthesis from protected amino acids contained in the analogues which are coupled in sequence and correspond to the amino acids in the analogues and which are optionally supplemented with corresponding amino acids which do not occur in the natural exendin peptides.
The glycine at position 30 of the exendin-3 or exendin-4 sequence was substituted by another proteogenic or non-proteogenic amino acid in order to avoid diketopiperazine formation during the synthesis after cleavage of the amino terminal protective group.
The exendin-(1-30) analogues and fragments are advantageous compared to the exendins-1-(1-39) since the shorter sequences of these analogues enable a more simple synthesis in higher yields.
The abbreviations and definitions of the amino acids that are used were recommended in Pure Appl. Chem. 31, 639-45 (1972) and ibid. 40, 277-90 (1974) and correspond to the PCT rules (WIPO standard st. 23:Recommendation for the Presentation of Nucleotide and Amino Acid Sequences in Patent Applications and in Published Patent Documents). The one and three letter codes are as follows:
The abbreviations represent L-amino acids if not specified otherwise such as D- or D,L-. The D-amino acids are written in small letters in the one letter code. Certain natural as well as non-natural amino acids are achiral e.g. glycine. In the representation the N-terminal end of all peptides is on the left and the C-terminal end is on the right.
Examples of non-proteinogenic amino acids are given in the following list together with their abbreviations:
All amino acids can be divided into the following three main classes according to their physical-chemical properties:
Acidic: The amino acid releases a proton in aqueous solution and at physiological pH and consequently carries a negative charge.
Basic: The amino acid accepts a proton in aqueous solution and at physiological pH and consequently carries a positive charge.
Neutral: The amino acid is in an uncharged state in aqueous solution and at physiological pH.
The definition xe2x80x9ccarries a positive/negative chargexe2x80x9d or xe2x80x9cis in an uncharged statexe2x80x9d only applies when on statistical average a significant number of a class of amino acids (at least 25%) are in the state.
In addition to the 20 so-called proteinogenic amino acids whose incorporation into proteins is controlled by the information of the genetic code, non-proteinogenic amino acids can also be incorporated into peptide sequences by the described synthesis process. A list of the proteinogenic amino acids and their classification into the above-mentioned three classes is given in Table 1. Non-proteinogenic amino acids are not genetically coded. Examples of non-proteinogenic amino acids and their classification into acidic, basic or neutral amino acids is given in Table 1.
The exendin analogoues which are a subject matter of the invention have advantageous therapeutic properties. Hence they lead to a stimulation of insulin release from the endocrine pancreas, to an increase of insulin biosynthesis and to increased peripheral glucose utilization. Since these effects can only be observed when the blood sugar levels are at the same time increased, a hypoglycemia would not be expected to occur after their administration. Furthermore the exendin analogues inhibit glucagon release from the endocrine pancreas and lead to a decrease of gluconeogenesis. In non-insulin dependent diabetes mellitus (NIDDM) the exendin analogues result in a considerable improvement of the metabolic situation. In particular the glucose uptake in muscle and fat tissue is increased independently of the insulin secretory effect. Due to the inhibitory effect on glucagon release, it is also appropriate to administer the exendin analogues in insulin dependent diabetes mellitus. Compared to glucagon-like peptide 1 (GLP-1) and the known exendin-3 and exendin-4 sequences, the exendin analogues according to the invention surprisingly have a higher efficacy in the various test systems so that they are more suitable for a therapeutic application than GLP-1, exendin-3 or exendin-4. The advantages of the new exendin analogues are in particular as follows: higher stability towards degradation and metabolism, longer duration of action, effectiveness at lower doses. Analogues based on exendin-3 are particularly preferred which exhibit particularly long durations of action or effectiveness at particularly low doses.
Solid phase and liquid phase synthesis is a conventional process for synthesizing peptides. In order to optimize the process for the synthesis of a particular product with regard to the purity of the crude product and yield, it is necessary that the process parameters and the materials that are used, for example the support material, the reagents which should make groups react, the materials for blocking the groups which should not react or the reagents which cleave blocking materials are adapted to the product to be synthesized, to the intermediate products to be synthesized and the starting materials. This adaptation is not simple with regard to the interdependency of the many process parameters.
Pharmaceutical preparations which contain the peptides according to the invention individually or together as an active substance in addition to conventional auxiliary substances and additives are preferably administered parenterally (subcutaneously, intramuscularly or intravenously). However, all other common forms of administration such as oral, rectal, buccal (including sublingual), pulmonary, transdermal, iontophoratic, vaginal and intranasal administration come into consideration. The drug has an insulin-regulating effect thereby promoting in an advantageous manner the compensation of the blood sugar level. It is advantageous for the use of the drug when blood levels between 20 and 50 pmol/l are attained. Infusion rates of 0.4-1.2 pmol/kg/min are necessary for this. In the case of a subcutaneous or buccal administration, substance quantities of 5-500 nmol are necessary depending on the galenic form and intended duration of action.
The exendin analogues according to the invention or pharmacologically acceptable salts thereof are preferably stored as sterile lyophilisates and mixed with a suitable isotonic solution before administration. The analogues can then be injected, infused or optionally also absorbed through the mucous membranes in this form. The conventional isotonic aqueous systems that are suitable for injection or infusion which contain common additives for injection solutions such as stabilizers and solubilizers can be used as solvents. Physiological saline solution or optionally solutions made isotonic by buffers are preferred in this case.
Additives are, for example, tartrate or citrate buffer, ethanol, complexing agents (such as ethylene diamintetraacetic acid and non-toxic salts thereof), high molecular polymers (such as liquid polyethylene oxide) to regulate the viscosity. Liquid carrier substances for injection solutions must be sterile and are preferably filled into ampoules. Solid carrier substances are for example starch, lactose, mannitol, methyl cellulose, talcum, highly dispersed silicic acids, higher molecular weight fatty acids (such as stearic acid), gelatin, agar-agar, calcium phosphate, magnesium stearate, animal or vegetable fats, solid high molecular polymers (such as polyethylene glycol); suitable preparations for oral administration can if desired contain flavourings or sweeteners. For nasal administration surfactants can be added to improve absorption through the nasal mucous membrane e.g. cholic acid, taurocholic acid, chenodeoxycholic acid, glycolic acid, dehydrocholic acid, deoxycholic acid and cyclodextrins.
The daily dose to be administered is in a range of 150-500 nmol. The determination of the biological activity is based on measurements compared to international reference preparations for glucagon-like peptide-1, exendin-3 or exendin-4 in a conventional test procedure for glucagon-like peptide-1.
The exendin analogues according to the invention can be prepared by conventional processes in peptide synthesis as described for example in J. M. Steward and J. D. Young xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984) and in J. Meienhofer Hormonal Proteins and Peptides, Vol.2 Academic Press, New York (1973) for solid phase synthesis and in E. Schroder and K. Lubke xe2x80x9cThe Peptidesxe2x80x9d, Vol.1, Academic Press, New York (1965) for liquid phase synthesis.
In general protected amino acids are added to a growing peptide chain for the synthesis of peptides. Either the amino group or the carboxyl group as well as any reactive group in the side chain of the first amino acid are protected. This protected amino acid is either coupled to an inert support or it can also be used in solution. The next amino acid in the peptide sequence is appropriately protected under conditions which favour the formation of an amide bond and is added to the first. After all desired amino acids have been coupled in the correct sequence, the protective groups and optionally the support phase are cleaved. The crude polypeptide that is obtained is reprecipitated and preferably purified chromatographically to form the final product.
A preferred method for synthesizing analogues of physiologically active polypeptides with fever than fourty amino acids comprises a solid phase peptide synthesis. In this method the xcex1-amino functions (Nxcex1) and any reactive side chains are protected with acid-labile or base-labile groups. The protective groups that are used should be stable under the conditions for linking amide bonds but it should be possible to readily cleave them without impairing the polypeptide chain that has formed. Suitable protective groups for the xcex1-amino function include the following groups but are not limited to these: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl, tert.-amyloxycarbonyl (Amoc), xcex1,xcex1-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl, 2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), 1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like. 9-Fluorenylmethoxycarbonyl (Fmoc) is preferably used as the Nxcex1-protective group.
Suitable side chain protective groups include the following but are not limited to these: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl, 2-chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl, 4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr), 2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl, tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl, trimethylsilyl and trityl (Trt).
In the solid phase synthesis the C-terminal amino acid is coupled as the first to a suitable support material. Suitable support materials are those which are inert towards the reagents and reaction conditions for the stepwise condensation and cleavage reactions and which do not dissolve in the reaction media that are used. Examples of commercially available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol and also chloromethylated styrene/divinylbenzene copolymers, hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers and the like. Polystyrene (1%)-divinylbenzene or TentaGel(copyright) (Rapp Polymere, Txc3xcbingen) derivatized with 4-benzyloxybenzyl-alcohol (Wang-anchor (Wang, S. S. 1973)) or 2-chlorotrityl chloride (Barlos, K. et al. 1989) is preferably used if it is intended to prepare the peptidic acid. In the case of the peptide amide, polystyrene (1%) divinylbenzene or TentaGel(copyright) derivatized with 5-(4xe2x80x2-aminomethyl)-3xe2x80x2,5xe2x80x2-dimethoxyphenoxy)valeric acid (PAL-anchor) (Albericio, F. et al. 1987) or p-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rink-Amid anchor (Rink, H. 1987)) is preferred.
The linkage to the polymeric support can be achieved by reacting the C-terminal Fmoc-protected amino acid with the support material with the addition of an activation reagent in ethanol, acetonitrile, N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents preferably in DMF at room temperature or elevated temperatures e.g. between 40xc2x0 C. and 60xc2x0 C., preferably at room temperature and with reaction times of 2 to 72 hours, preferably about 2xc3x972 hours.
The coupling of the Nxcex1 protected amino acid preferably the Fmoc amino acid to the PAL, Wang or Rink anchor can for example be carried out with the aid of coupling reagents such as N,Nxe2x80x2-dicyclohexylcarbodiimide (DCC), N,Nxe2x80x2-diisopropylcarbodiimide (DIC) or other carbodiimides, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or other uronium salts, o-acyl-ureas, benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimides, other N-hydroxyimides or oximes in the presence or also in the absence of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole preferably with the aid of TBTU with addition of HOBt, with or without the addition of a base such as for example diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine, preferably diisopropylethylamine with reaction times of 2 to 72 hours, preferably 3 hours in a 1.5 to 3-fold excess of the amino acid and the coupling reagents, preferably in a 2-fold excess and at temperatures between about 10xc2x0 C. and 50xc2x0 C., preferably 25xc2x0 C. in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, preferably dimethylformamide. Instead of the coupling reagents it is also possible to use the active esters (e.g. pentafluorophenyl, p-nitrophenyl or the like), the symmetric anhydride of the Nxcex1-Fmoc-amino acid, its acid chloride or acid fluoride under the conditions described above.
The Nxcex1-protected amino acid, preferably the Fmoc amino acid is preferably coupled to the 2-chlorotrityl resin in dichloromethane with the addition of DIEA with reaction times of 10 to 120 minutes, preferably 20 minutes but is not limited to the use of this solvent and this base.
The successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis typically in an automated peptide synthesizer. After cleavage of the Nxcex1-Fmoc protective group of the coupled amino acid on the solid phase by treatment with piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes, preferably 2xc3x972 minutes with 50% piperidine in DMF and 1xc3x9715 minutes with 20% piperidine in DMF, the next protected amino acid in a 3 to 10-fold excess, preferably in a 10-fold excess is coupled to the previous amino acid in an inert, non-aqueous, polar solvent such as dichloromethane, DMF or mixtures of the two, preferably DMF and at temperatures between about 10xc2x0 C. and 50xc2x0 C., preferably at 25xc2x0 C. The reagents that have already been mentioned for coupling the first Nxcex1-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable as coupling reagents. Active esters of the protected amino acid, or chlorides or fluorides or symmetric anhydrides thereof can also be used as an alternative.
At the end of the solid phase synthesis the peptide is cleaved from the support material while simultaneously cleaving the side chain protecting groups. Cleavage can be carried out with trifluoroacetic acid or other strongly acidic media with addition of 5%-20% V/V scavengers such as dimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol, anisole ethanedithiol, phenol or water, preferably 15% v/v dimethylsulfide/ethanedithiol/m-cresol 1:1:1 within 0.5 to 3 hours, preferably 2 hours. Peptides with fully protected side chains are obtained by cleaving the 2-chlorotrityl anchor with glacial acetic acid/trifluoroethanol/dichloromethane 2:2:6. The protected peptide can be purified by chromatography on silica gel. If the peptide is linked to the solid phase via the Wang anchor and if it is intended to obtain a peptide with a C-terminal alkylamidation, the cleavage can be carried out by aminolysis with an alkylamine or fluoroalkylamine. The aminolysis is carried out at temperatures between about xe2x88x9210xc2x0 C. and 50xc2x0 C., preferably about 25xc2x0 C. and reaction times between about 12 and 24 hours, preferably about 18 hours. In addition the peptide can also be cleaved from the support by re-esterification e.g. with methanol.
The acidic solution that is obtained is admixed with a 3 to 20-fold amount of cold ether or n-hexane, preferably a 10-fold excess of diethyl ether, in order to precipitate the peptide and hence to separate the scavengers and cleaved protective groups that remain in the ether. A further purification can be carried out by re-procipitating the peptide several times from glacial acetic acid. The precipitate that is obtained is taken up in water or tert. butanol or mixtures of the two solvents, preferably a 1:1 mixture of tert, -butanol/water and freeze-dried.
The peptide obtained can be purified by some or all of the following chromatographic methods: ion exchange over a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on non-derivatized polystyrene/divinylbenzene copolymers (e.g. Amberlite(copyright) XAD); adsorption chromatography on silica gel; ion exchange chromatography e.g. on carboxymethyl cellulose; distribution chromatography e.g. on Sephadex(copyright) G-25; countercurrent distribution chromatography; or high pressure liquid chromatography (HPLC) in particular reversed-phase HPLC on octyl or octadecylsilylsilica (ODS) phases.
In summary part of the present invention encompasses processes for the preparation of polypeptides and pharmaceutically usable salts thereof. These processes which lead to physiologically active shortened homologues and analogues of exendin-3 or exendin-4 with the above-mentioned preferred chain lengths and modifications comprise processes for the sequential condensation of protected amino acids on a suitable support material, methods for cleaving the support and protective groups and for purifying the crude peptides that are obtained.
The amino acid analysis Was carried out with an amino acid analyzer 420 A from the Applied Biosystems Company (Weiterstadt). 50 to 1000 pmol of the sample to be analysed was applied to the sample carrier in 10 to 40 xcexcl solution and subsequently fully automatically hydrolysed for 90 minutes in the gas phase at 160xc2x0 C. with 6 N hydrochloric acid, derivatized with phenylisothiocyanate and analysed on-line by a microbore HPLC. Mass spectroscopic examinations were carried out on an API III triple-quadrupole mass spectrometer (SCIEX; Thornhill, Canada) equipped with an ion spray ion source.
The protected amino acid derivatives can for example be obtained from Novabiochem GmbH (Bad Soden).